PROJECT SUMMARY B cell tolerance is key to the development of an immune system tolerant to self, but the mechanisms that enforce this process are not yet clearly understood. The overarching goal of our studies is to elucidate the molecular pathways that regulate B cell selection at the developmental checkpoint between the immature and transitional cell stages, enforcing the process of tolerance in autoreactive bone marrow B cells while promoting the differentiation of nonautoreactive cells and their bone marrow exit. By studying Ig knock-in mouse models of central B cell tolerance we documented that the RAS, ERK, and PI3K signaling molecules, which are GTPases, MAP kinases, and lipid kinases respectively, are active to a higher basal level in nonautoreactive than autoreactive immature B cells. Our studies have found that while both ERK and PI3K are necessary for the in vitro differentiation of immature B cells into transitional B cells, only the in vivo activation of PI3K in B cells, but not that of ERK 1) abrogates central B cell tolerance, including receptor editing and clonal deletion, 2) supports the exit of autoreactive B cells from the bone marrow, and 3) promotes their further maturation and activation. Taken together, these findings establish that the PI3K pathway can override central and early peripheral tolerance in mouse autoreactive B cells, even when these cells possess high avidity for the self- antigen. The goal of this grant is to define the mechanisms by which PI3K controls this central checkpoint of B cell selection in mice and whether it similarly functions in human B cells. Experiments described in Aims 1 and 2 will utilize mouse models of tolerance to investigate whether and how FOXO1, CXCR4, and ERK translate PI3K function in immature B cells. RNAseq analyses of nonautoreactive B cells and of autoreactive B cells that either break or make tolerance will aid the discovery of additional PI3K mediators in this process. Patients with gain-of-function mutations in PI3K? have been recently discovered, and they often exhibit autoantibodies and other autoimmune manifestations. Experiments in Aim 3 will utilize two complementary and novel approaches to determine the ability of gain-of-function mutations in PI3K? to break central tolerance in human B cells. Overall, these studies are significant because they will generate a deeper mechanistic understanding of how the primary B cell repertoire is formed and how B cell tolerance can be breached during the development of autoreactive B cells, raising the autoreactive capacity of the primary B cell repertoire and, consequently, the risk for autoimmunity.